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In Silico Improvement of beta(3)-Peptide Inhibitors of p53 centerdot hDM2 and p53 center dot hDMX

Citation for published version:Michel, J, Harker, EA, Tirado-Rives, J, Jorgensen, WL & Schepartz, A 2009, 'In Silico Improvement ofbeta(3)-Peptide Inhibitors of p53 center dot hDM2 and p53 center dot hDMX', Journal of the AmericanChemical Society, vol. 131, no. 18, pp. 6356–6357. https://doi.org/10.1021/ja901478e

Digital Object Identifier (DOI):10.1021/ja901478e

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Published In:Journal of the American Chemical Society

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Download date: 16. Jun. 2020

In silico improvement of β3-peptide inhibitors of p53•hDM2 andp53•hDMX

Julien Michel, Elizabeth A. Harker, Julian Tirado-Rives, William L. Jorgensen*, and AlannaSchepartz*Departments of Chemistry and Molecular, Cellular, and Developmental Biology, Yale University,New Haven, Connecticut 06520-8107

AbstractThere is great interest in molecules capable of inhibiting the interactions between p53 and its negativeregulators hDM2 and hDMX, as these molecules have validated potential against cancers in whichone or both oncoproteins are overexpressed. We reported previously that appropriately substitutedβ3-peptides inhibit these interactions and, more recently, that minimally cationic β3-peptides aresufficiently cell permeable to upregulate p53-dependent genes in live cells. These observations,coupled with the known stability of β-peptides in a cellular environment, and the recently reportedstructures of hDM2 and hDMX, motivated us to exploit computational modeling to identify β-peptides with improved potency and/or selectivity. This exercise successfully identified a new β3-peptide, β53-16, that possesses the highly desirable attribute of high affinity for both hDM2 as wellas hDMX and identifies the 3,4-dichlorophenyl moiety as a novel determinant of hDMX affinity.

Email: william.jorgensen@yale.edu, alanna.schepartz@yale.edu.Supporting Information Available: Details of computational protocols, CD and binding assays and the complete citation for reference20. This material is available free of charge via the Internet at http://pubs.acs.org

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Published in final edited form as:J Am Chem Soc. 2009 May 13; 131(18): 6356–6357. doi:10.1021/ja901478e.

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There is great interest in molecules that inhibit interactions between p53 and its negativeregulators hDM2 and hDMX, as these molecules have validated potential against cancers thatoverexpress one or both of these oncoproteins.1,2 We reported that substituted β3-peptides caninhibit these interactions3,4 and, more recently, that minimally cationic β3-peptides aresufficiently cell permeable to upregulate p53-dependent genes in live cells.5,6 Theseobservations, coupled with the established intracellular stability of β-peptides7–9 and therecently reported structures of hDM210 and hDMX,11 motivated us to exploit computationalmethods to identify β-peptides with improved potency and/or selectivity. This exercisesuccessfully identified a new β3-peptide, β53-16, that possesses the desirable attribute of highaffinity for hDM2 and hDMX and identifies the 3,4-dichlorophenyl moiety as a noveldeterminant of hDMX affinity.

Our computational modeling began with the application of Visual Molecular Dynamics (VMD)12 to generate a model of previously reported β53-8 bound to the p53 binding site on hDM2(Figure 1A). In this model, β53-8 is bound as a 14-helix that is slightly unwound at the C-terminus, mimicking its conformation in solution.13 The three hDM2 hydrophobic pocketsoccupied in the native structure by the p53 side chains of Leu26, Trp23 and Phe19 10 are occupiedin the modeled complex by the corresponding β3-amino acid side chains at positions 3, 6, and9. An analogous model of β53-8 bound to hDMX was also prepared (Figure 1B).11

We then applied a hierarchical computational strategy to search for alternative side chains thatwould improve packing at one or both interfaces. With the de novo design programBOMB14 we screened over ten thousand β53-8 analogs containing substituted aromatic andnon-aromatic heterocycles and short hydrocarbon side chains in place of Leu26, Trp23 andPhe19.10 About 50 candidates were identified by scoring and visualization for evaluation withMCPRO.15 Binding free energies were predicted via Monte Carlo Free Energy Perturbation(MC/FEP) calculations using the OPLS-AA force field16 for the protein-ligand complex andthe TIP4P model for water.17 In these simulations, the protein backbones remained fixed; the

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affinities of the eight most interesting and synthetically accessible compounds (Figure 1C)were subsequently reevaluated in a second round of MC/FEP calculations that permittedbackbone motions.18

The models were first validated by evaluating whether they would predict the large increasein hDM2 affinity realized when the tryptophan side chain at position 6 is replaced by 6-chlorotryptophan (6-ClW) (compare β53-8 and β53-13,Figure 1C).19 The calculations predictthat substitution of 6-ClW at position 6 should significantly improve binding to hDM2 (ΔΔG= –2.1 kcal•mol−1) but not hDMX (ΔΔG = +1.0 kcal•mol−1,Figure 2C). These predictions arefully aligned with the experimental results: the stability of the hDM2•β53-13 complex issignificantly higher (Kd = 30.1 nM, ΔG = –10.25 kcal•mol−1) than that of the hDM2•β53-8complex (Kd = 204 nM, ΔG = –9.12 kcal•mol−1), whereas the stabilities of the analogoushDMX complexes are comparable (Kd = 1.6 and 2.1 µM for β53-13 and β53-8, respectively).The improvement in hDM2 but not hDMX affinity upon substitution of 6-ClW is consistentwith results observed in the context of previously reported ligands.20–23

The models were further validated by their ability to predict the large increase in hDM2 andhDMX affinity observed for β-peptides containing a central meta-trifluoromethyl phenylsubstituent (CF3F) when compared with an unsubstituted phenyl ring (compare β53-12 withβ53-14,Figure 1C). The calculations predict that the CF3F side chain should favor binding toboth hDM2 and hDMX (ΔΔG = –4.8 and –4.6 kcal•mol−1, respectively). This increase wasalso realized experimentally, albeit in an attenuated way: the stability of the hDM2•β53-12complex is significantly higher (Kd = 28.2 nM, ΔG = –10.29 kcal•mol−1) than that of thehDM2•β53-14 complex (Kd = 816 nM, ΔG = –8.3 kcal•mol−1); analogous differences are seenfor the hDMX complexes (Figure 2).24

Next we examined whether the affinity of β53-12 could be increased further by substitutingthe leucine side chain at position 6 with one of eight cyclic and acyclic hydrocarbonalternatives. Although few promising candidates emerged from the BOMB and MC/FEPanalyses, we did investigate β53-17, in which the Leu side chain is replaced by Ile. Thissubstitution was predicted to slightly favor the binding of both hDM2 and hMDX (ΔΔGbind =–0.9 and –0.3 kcal•mol−1, respectively). However, no increase in affinity was observed, andthese molecules were not studied further. We note that 53-17 is significantly less 14-helicalthan 53-12 as judged by circular dichroism analysis (Figure SI-1). As the computational modeldoes not account for changes in β-peptide secondary structure, it is possible that the observedchange in secondary structure accounts for the poor agreement between prediction andexperiment in this case. The predictions may be also be affected by uncertainty in the structuresof unliganded hDM2 and hDMX as the 23 N-terminal residues of both proteins are onlypartially resolved due to their flexibility.25,26

Based on these observations, we returned attention to the central side chain of the hDM2/hDMXepitope and evaluated the relative hDM2 and hDMX affinities of hundreds of β53-12 analogscontaining substituted phenylalanine analogs at position 6. This analysis suggested that β-peptides containing either meta-chlorophenylalanine or para-chlorophenylalanine at thisposition would show improved affinity for both hDM2 and hDMX when compared withβ53-14 (−3.5 kcal•mol−1 > ΔΔGbind > −2.5 kcal•mol−1). Indeed, the stability of thehDM2•β53-15 complex (meta-chloro substituent,Figure 1C) is significantly higher (Kd = 150nM, ΔG = −9.3 kcal•mol−1) than that of hDM2·β53-14; analogous differences are observedfor the hDMX complexes (Figure 2). However, as predicted, the stabilities of the β53-15complexes were not greater than those of the β53-12 complexes. Therefore, since the gains inaffinity for the para-chlorophenylalanine were predicted to be similar to those of 53-15, thisadditional analog was not tested experimentally.

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Finally we examined the effect of a meta,para-dichlorophenylalanine side chain at the centralposition of the recognition epitope (β53-16,Figure 1), whose inclusion was predicted tosignificantly improve affinity for both hDM2 and hDMX compared to β53-14 (ΔΔGbind = −4.4and −5.4 kcal•mol−1, respectively. Indeed, the stabilities of both hDM2•β53-16 andhDMX•β53-16 are significantly higher than the corresponding β53-14 complexes (Kd = 27.6nM and 155 nM, respectively for β53-16,Figure 2). They also equal or exceed the stabilitiesof the corresponding complexes with 53-12. Competition fluorescence polarizationexperiments confirm that β53-16 competes with p53AD for binding to hDM2 and hDMX andshows improved inhibitory potency towards hDMX (Figure SI-2).

Thus, β53-16 offers significantly improved affinity for hDMX without loss of affinity forhDM2. Analysis of the MC/FEP simulations suggests more favorable interaction of thedichlorophenyl group with residues 50–54 in hDMX than with equivalent residues 54–58 inhDM2. We subsequently examined whether the affinity of β53-16 could be improved furtherupon replacement of the adjacent phenylalanine side chain with one of twelve substitutedanalogs. This scan failed to identify promising substitutions as the phenylalanine side chainappears to bind tightly to the hydrophobic pocket of both proteins; however minor gains inaffinity for both hDM2 and hDMX were predicted for a para-fluorophenylalanine substitution(ΔΔGbind = −0.4 and −0.9 kcal•mol−1, respectively). No increase in affinity was observedexperimentally with this peptide, β53-18, so further modification at this position was notpursued (Figure 1, Table 1). β53-16 represents the highest affinity β3-peptide for hDMXreported to date, with significantly higher affinity than the prototypic hDM2 ligand, Nutlin-3.Thus, β53-16 embodies the pan-specificity of well known peptidic hDM2/hDMXinhibitors27,28 without the limitations of protease sensitivity or poor uptake.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentThis work was supported by the NIH (GM 74756 and GM 32136) and the National Foundation for Cancer Research.

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Figure 1.Computationally generated models of β53-8 (blue) in complex with (a) hDM2 and (b) hDMXillustrating differences in binding site topologies. (c) Helical net representations of β3-peptidesstudied herein.

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Figure 2.Direct fluorescence polarization analysis of the affinity of each β-peptide shown for (A) hDM2and (B) hDMX. (C) Comparison of calculated and experimental binding free energiesexpressed in terms of ΔΔGbind relative to the standard shown (kcal•mol−1); Kd values in nMunits.

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